PROJECT SUMMARY Because the heart has negligible intrinsic capacity to regenerate new tissues to replace those lost to injury, there is currently no definitive heart failure treatment, other than organ transplantation. Recent studies have introduced the prospect of replacing damaged heart tissues with healthy cardiomyocytes derived from pluripotent stem cells. However, realizing the full therapeutic potential of stem cells faces numerous hurdles, including the potential for tumor formation, a low rate of cardiomyocyte formation, and an inadequate mechanistic understanding of cardiomyogenesis. Additionally, translational efforts are hampered by a lack of pharmaceutical agents to boost therapeutic effects of stem cells. Dorsomorphin, the first known small molecule inhibitor of the bone morphogenetic protein (BMP) signaling, is one of the most potent chemical inducers of cardiomyogenesis in mouse embryonic stem (ES) cells. Dorsomorphin treatment during the initial 24 to 48 hours of ES cell differentiation was sufficient for robust cardiomyocyte induction. Strikingly, the massive cardiac induction occurs apparently in the absence of mesoderm induction and at the expense of other mesoderm-derived lineages, including endothelial, smooth muscle and hematopoietic lineages. From these results, we hypothesize that a timely BMP signal inhibition commits the primitive multipotent progenitor cells toward the cardiomyocyte development. The present study takes advantage of this unique and powerful model of cardiac induction to elucidate the mechanism of cardiomyogenic commitment and differentiation. In Aim 1, we examine in detail the effects of small molecule BMP inhibitors on mesoderm, cardiovascular progenitor and cardiomyocyte formation. By comparing the effects of dorsomorphin and an exclusively specific BMP inhibitor DMH1, we will test whether dorsomorphin's known off-target effects against the Flk1 kinase reduces overall mesoderm formation, and whether a pure BMP inhibitor could induce even greater cardiomyocyte formation. In Aim 2, we will examine whether the Flk1+ mesoderm progenitor cells from DMH1-treated ES cells are preferentially committed to become cardiomyocytes. In addition, we will determine the molecular profile of the putative cardiac-committed progenitors induced by the DMH1 treatment. These studies will shed much needed light on the nature of cardiac progenitor cells. In Aim 3, we will take the next logical step to test whether small molecules that robustly induce cardiomyocyte formation in vitro can have a beneficial impact on stem cell therapies to improve cardiac repair in a mouse model of myocardial injury. Utilizing the unique ability of small molecules to block BMP signaling in adult animals, we will explore the potential of in vivo stimulation of cardiomyocyte formation following ES cell transplantation. The present study, which utilizes the small molecule BMP inhibitors to probe the mechanism of cardiomyogenesis, will not only inform future stem cell-based strategies to treat heart disease, but provide valuable pharmaceutical agents to boost the therapeutic effects of stem cells.